Important Plant Pathogens Research Articles

Bacterial translation machinery for deliberate mistranslation of the genetic code

Inaccurate expression of the genetic code, also known as mistranslation, is an emerging paradigm in microbial studies. Growing evidence suggests that many microbial pathogens can deliberately mistranslate their genetic code to help invade a host or evade host immune responses. However, discovering different capacities for deliberate mistranslation remains a challenge because each group of pathogens typically employs a unique mistranslation mechanism. In this study, we address this problem by studying duplicated genes of aminoacyl-transfer RNA (tRNA) synthetases. Using bacterial prolyl-tRNA synthetase (ProRS) genes as an example, we identify an anomalous ProRS isoform, ProRSx, and a corresponding tRNA, tRNAProA, that are predominately found in plant pathogens from Streptomyces species. We then show that tRNAProA has an unusual hybrid structure that allows this tRNA to mistranslate alanine codons as proline. Finally, we provide biochemical, genetic, and mass spectrometric evidence that cells which express ProRSx and tRNAProA can translate GCU alanine codons as both alanine and proline. This dual use of alanine codons creates a hidden proteome diversity due to stochastic Ala→Pro mutations in protein sequences. Thus, we show that important plant pathogens are equipped with a tool to alter the identity of their sense codons. This finding reveals the initial example of a natural tRNA synthetase/tRNA pair for dedicated mistranslation of sense codons.

Increased abundance of secreted hydrolytic enzymes and secondary metabolite gene clusters define the genomes of latent plant pathogens in the Botryosphaeriaceae

BackgroundThe Botryosphaeriaceae are important plant pathogens, but also have the ability to establish asymptomatic infections that persist for extended periods in a latent state. In this study, we used comparative genome analyses to shed light on the genetic basis of the interactions of these fungi with their plant hosts. For this purpose, we characterised secreted hydrolytic enzymes, secondary metabolite biosynthetic gene clusters and general trends in genomic architecture using all available Botryosphaeriaceae genomes, and selected Dothideomycetes genomes.ResultsThe Botryosphaeriaceae genomes were rich in carbohydrate-active enzymes (CAZymes), proteases, lipases and secondary metabolic biosynthetic gene clusters (BGCs) compared to other Dothideomycete genomes. The genomes of Botryosphaeria, Macrophomina, Lasiodiplodia and Neofusicoccum, in particular, had gene expansions of the major constituents of the secretome, notably CAZymes involved in plant cell wall degradation. The Botryosphaeriaceae genomes were shown to have moderate to high GC contents and most had low levels of repetitive DNA. The genomes were not compartmentalized based on gene and repeat densities, but genes of secreted enzymes were slightly more abundant in gene-sparse regions.ConclusionThe abundance of secreted hydrolytic enzymes and secondary metabolite BGCs in the genomes of Botryosphaeria, Macrophomina, Lasiodiplodia, and Neofusicoccum were similar to those in necrotrophic plant pathogens and some endophytes of woody plants. The results provide a foundation for comparative genomic analyses and hypotheses to explore the mechanisms underlying Botryosphaeriaceae host-plant interactions.

The "Bipartite" Structure of the First Genome of Ampelomyces quisqualis, a Common Hyperparasite and Biocontrol Agent of Powdery Mildews, May Point to Its Evolutionary Origin from Plant Pathogenic Fungi.

Powdery mildews are among the most important plant pathogens worldwide, which are often attacked in the field by mycoparasitic fungi belonging to the genus Ampelomyces. The taxonomy of the genus Ampelomyces is unresolved, but well-supported molecular operational taxonomic units were repeatedly defined suggesting that the genus may include at least four to seven species. Some Ampelomyces strains were commercialized as biocontrol agents of crop pathogenic powdery mildews. However, the genomic mechanisms underlying their mycoparasitism are still poorly understood. To date, the draft genome of a single Ampelomyces strain, designated as HMLAC 05119, has been released. We report a high-quality, annotated hybrid draft genome assembly of A. quisqualis strain BRIP 72107, which, based on phylogenetic analyses, is not conspecific with HMLAC 05119. The constructed genome is 40.38 Mb in size, consisting of 24 scaffolds with an N50 of 2.99 Mb and 96.2% completeness. Our analyses revealed “bipartite” structure of Ampelomyces genomes, where GC-balanced genomic regions are interspersed by longer or shorter stretches of AT-rich regions. This is also a hallmark of many plant pathogenic fungi and provides further evidence for evolutionary affinity of Ampelomyces species to plant pathogenic fungi. The high-quality genome and annotation produced here provide an important resource for future genomic studies of mycoparasitisim to decipher molecular mechanisms underlying biocontrol processes and natural tritrophic interactions.

Transcript pattern analysis of Arf-family genes in the phytopathogen Fusarium oxysporum f. sp. lycopersici reveals the role of Arl3 in the virulence.

Fusarium oxysporum f. sp. lycopersici is an important plant pathogen that has been used to understand the virulence mechanisms that soil inhabiting fungi exhibit during the infection process. In F. oxysporum many of the virulence factors are secreted, and the secretion process requires the formation of vesicles. Arf family members, represented by Arf (ADP- Ribosylation Factor), Arl (Arf-like), and Sar (Secretion-associated and Ras-related) proteins, are involved in the vesicle creation process. In this study we identified the Arf family members in F. oxysporum f. sp. lycopersici, which includes seven putative proteins: Arf1, Arf3, Arl1 through Arl3, Arl8B, and Sar1. Quantification of the mRNA levels of each arf encoding gene revealed that the highest expression corresponds to arf1 in all tested conditions. The phylogenetic analysis revealed that no other Arf1 paralogue, such as Arf2 from yeast, is present in F. oxysporum f. sp. lycopersici. The essential function suggested of Arf1 in F. oxysporum f. sp. lycopersici was corroborated experimentally when, after several attempts, it was impossible to obtain a knockout mutant in arf1. Moreover, arl3 mRNA levels increased significantly when plant tissue was added as a sole carbon source, suggesting that the product of these genes could play pivotal roles during plant infection, the corresponding mutant ∆arl3 was less virulent compared to the wild-type strain. These results describe the role of arl3 as acritical regulator of the virulence in F. oxysporum f. sp. lycopersici and stablish a framework for the arf family members to be studied in deeper details in this phytopathogen.

Foliar selenium application reduces late blight severity and incidence in potato and acts as a pathogen growth inhibitor and elicitor of induced plant defence

Late blight caused by Phytophthora infestans is a serious threat to potato production. Currently, solutions to manage late blight in organic systems are scarce. This study was undertaken to evaluate the effect of selenium (Se) on plant emergence from seed pre-treated with Se and the seed decay pathogens Pectobacterium carotovorum ssp. carotovorum and Fusarium sambucinum, and on foliar potato late blight following inoculation with P. infestans and foliar treatment with Se. The potential modes of action of Se were also evaluated. Following inoculation of the seed decay pathogens on potato seed pre-treated with different rates of Se, plant emergence was not affected by Se treatment. By pre-treating potato seed with different Se rates, and applying different foliar Se rates on potato plants subsequently inoculated with the late blight pathogen in a greenhouse setting, we showed that foliar Se application alone or combined with Se seed treatment reduced the severity and incidence of late blight, and activated the production of secondary metabolites in potato leaves and tubers. Further, in vitro studies showed that Se directly inhibited the mycelial growth of P. infestans and other important plant pathogens, including Fusarium graminearum and Sclerotinia sclerotiorum. The data suggest that Se acts as an inducer of plant defences, while also inhibiting fungal growth. The study highlights the potential of Se for managing late blight, and suggests that its application could contribute to improving plant health in greenhouse production systems by helping to control diseases caused by P. infestans, F. graminearum and S. sclerotiorum.

Implication of the Type III Effector RipS1 in the Cool-Virulence of Ralstonia solanacearum Strain UW551.

Members of the Ralstonia solanacearum species complex cause a variety of wilting diseases across a wide range of hosts by colonizing and blocking xylem vessels. Of great concern are race 3 biovar 2 strains of R. solanacearum capable of causing brown rot of potato at cool temperatures, which are select agents in the United States. To gain a better understanding of cool-virulence mechanisms, we generated libraries of transposon mutants in the cool-virulent R. solanacearum strain UW551 and screened 10,000 mutants using our seedling assay for significantly reduced virulence at 20°C. We found several mutants that exhibited reduced virulence at 28 and 20°C and also mutants that were only affected at the cooler temperature. One mutant of the latter chosen for further study had the transposon inserted in an intergenic region between a type III secretion system effector gene ripS1 and a major facilitator superfamily (MFS) protein gene. Gene expression analysis showed that expression of ripS1 was altered by the transposon insertion, but not the MFS protein gene. An independent mutant with this insertion upstream of ripS1 was generated and used to confirm virulence and gene expression phenotypes. The effector, RipS1, has unknown function and is part of a family of effectors belonging to the largest known type III effectors. The functional connection between RipS1 and cool-virulence of R. solanacearum UW551 suggests that RipS1 (and/or its upstream promoter element) may serve as a potential target for development of cool-virulence-specific diagnostic tools to differentiate the highly regulated cool-virulent strains from non-cool-virulent strains of R. solanacearum. Our results provide important information for continued work toward a better understanding of cool-virulence of R. solanacearum and development of proper control strategies to combat this important plant pathogen.

Microplastics accumulate fungal pathogens in terrestrial ecosystems

Microplastic (MP) is a pervasive pollutant in nature that is colonised by diverse groups of microbes, including potentially pathogenic species. Fungi have been largely neglected in this context, despite their affinity for plastics and their impact as pathogens. To unravel the role of MP as a carrier of fungal pathogens in terrestrial ecosystems and the immediate human environment, epiplastic mycobiomes from municipal plastic waste from Kenya were deciphered using ITS metabarcoding as well as a comprehensive meta-analysis, and visualised via scanning electron as well as confocal laser scanning microscopy. Metagenomic and microscopic findings provided complementary evidence that the terrestrial plastisphere is a suitable ecological niche for a variety of fungal organisms, including important animal and plant pathogens, which formed the plastisphere core mycobiome. We show that MPs serve as selective artificial microhabitats that not only attract distinct fungal communities, but also accumulate certain opportunistic human pathogens, such as cryptococcal and Phoma-like species. Therefore, MP must be regarded a persistent reservoir and potential vector for fungal pathogens in soil environments. Given the increasing amount of plastic waste in terrestrial ecosystems worldwide, this interrelation may have severe consequences for the trans-kingdom and multi-organismal epidemiology of fungal infections on a global scale.

Unravelling hybridization in Phytophthora using phylogenomics and genome size estimation

The genus Phytophthora comprises many economically and ecologically important plant pathogens. Hybrid species have previously been identified in at least six of the 12 phylogenetic clades. These hybrids can potentially infect a wider host range and display enhanced vigour compared to their progenitors. Phytophthora hybrids therefore pose a serious threat to agriculture as well as to natural ecosystems. Early and correct identification of hybrids is therefore essential for adequate plant protection but this is hampered by the limitations of morphological and traditional molecular methods. Identification of hybrids is also important in evolutionary studies as the positioning of hybrids in a phylogenetic tree can lead to suboptimal topologies. To improve the identification of hybrids we have combined genotyping-by-sequencing (GBS) and genome size estimation on a genus-wide collection of 614 Phytophthora isolates. Analyses based on locus- and allele counts and especially on the combination of species-specific loci and genome size estimations allowed us to confirm and characterize 27 previously described hybrid species and discover 16 new hybrid species. Our method was also valuable for species identification at an unprecedented resolution and further allowed correct naming of misidentified isolates. We used both a concatenation- and a coalescent-based phylogenomic method to construct a reliable phylogeny using the GBS data of 140 non-hybrid Phytophthora isolates. Hybrid species were subsequently connected to their progenitors in this phylogenetic tree. In this study we demonstrate the application of two validated techniques (GBS and flow cytometry) for relatively low cost but high resolution identification of hybrids and their phylogenetic relations.

The H4K20 methyltransferase Kmt5 is involved in secondary metabolism and stress response in phytopathogenic Fusarium species

Fusarium fujikuroi and Fusarium graminearum are agronomically important plant pathogens, both infecting important staple food plants and thus leading to huge economic losses worldwide. F.fujikuroi belongs to the Fusarium fujikuroi species complex (FFSC) and causes bakanae disease on rice, whereas F.graminearum, a member of the Fusarium graminearum species complex (FGSC), is the causal agent of Fusarium Head Blight (FHB) disease on wheat, barley and maize. In recent years, the importance of chromatin regulation became evident in the plant-pathogen interaction. Several processes, including posttranslational modifications of histones, have been described as regulators of virulence and the biosynthesis of secondary metabolites. In this study, we have functionally characterised methylation of lysine 20 histone 4 (H4K20me) in both Fusarium species. We identified the respective genes solely responsible for H4K20 mono-, di- and trimethylation in F.fujikuroi (FfKMT5) and F.graminearum (FgKMT5). We show that loss of Kmt5 affects colony growth in F.graminearum while this is not the case for F.fujikuroi. Similarly, FgKmt5 is required for full virulence in F.graminearum as Δfgkmt5 is hypovirulent on wheat, whereas the F.fujikuroi Δffkmt5 strain did not deviate from the wild type during rice infection. Lack of Kmt5 had distinct effects on the secondary metabolism in both plant pathogens with the most pronounced effects on fusarin biosynthesis in F.fujikuroi and zearalenone biosynthesis in F.graminearum. Next to this, loss of Kmt5 resulted in an increased tolerance towards oxidative and osmotic stress in both species.

Efficient genome editing using endogenous U6 snRNA promoter-driven CRISPR/Cas9 sgRNA in Sclerotinia sclerotiorum

We previously reported on a CRISPR-Cas9 genome editing system for the necrotrophic fungal plant pathogen Sclerotinia sclerotiorum. This system (the TrpC-sgRNA system), based on an RNA polymerase II (RNA Pol II) promoter (TrpC) to drive sgRNA transcription in vivo, was successful in creating gene insertion mutants. However, relatively low efficiency targeted gene editing hampered the application of this method for functional genomic research in S. sclerotiorum. To further optimize the CRISPR-Cas9 system, a plasmid-free Cas9 protein/sgRNA ribonucleoprotein (RNP)-mediated system (the RNP system) and a plasmid-based RNA polymerase III promoter (U6)-driven sgRNA transcription system (the U6-sgRNA system) were established and evaluated. The previously characterized oxaloacetate acetylhydrolase (Ssoah1) locus and a new locus encoding polyketide synthase12 (Sspks12) were targeted in this study to create loss-of-function mutants. The RNP system, similar to the TrpC-sgRNA system we previously reported, creates mutations at the Ssoah1 gene locus with comparable efficiency. However, neither system successfully generated mutations at the Sspks12 gene locus. The U6-sgRNA system exhibited a significantly higher efficiency of genemutation at both loci. This technology provides a simple and efficient strategy for targeted gene mutation and thereby will accelerating the pace of research of pathogenicity and development in this economically important plant pathogen.

Construction of the PG-deficient mutant of Fusarium equiseti by CRISPR/Cas9 and its pathogenicity of pitaya.

Fusarium is an important plant pathogen and many cell wall-degrading enzymes (CWDEs) are produced in Fusarium-infected plant tissues. To investigate the role of CWDEs in the pathogenicity of pitaya pathogen, we isolated a Fusarium equiseti strain from the diseased pitaya fruit and the activities of CWDEs were determined. The higher polygalacturonase (PG) activity was confirmed both in vitro and vivo. Aiming at the PG gene, the CRISPR/Cas9 system of F. equiseti was constructed and optimized for the first time. Through the process of microhomology-mediated end joining, the flanking region containing 30 bp was used to mediate the homologous recombination of Cas9 double-strand breaks, and the PG gene knockout mutants were obtained by protoplast transformation. Through the phenotypic and pathogenicity experiments of the wild-type strain and mutant strain, the results showed that the colony growth rate and spore production of the strain without the PG gene decreased to some extent, and the lesion diameter and the degree of pericarp cell damage decreased, which showed that the CRISPR/Cas9 system could be used in F. equiseti and PG enzyme and can play a significant role in the interaction between F. equiseti and pitaya fruit.

The Chromosome-Scale Genome Resource for Two Endophytic Fusarium species, F. culmorum and F. pseudograminearum.

Genus Fusarium (Ascomycota, Hypocreales, Nectriaceae) includes many economically important plant pathogens that cause devastating diseases of a wide range of crops and trees. Interestingly, there is increasing evidence that some Fusarium species also live as endophytes and benefit plant growth and stress tolerance. In this work, we sequence the whole genomes of endophytic F. culmorum and F. pseudograminearum, isolated from a coastal dunegrass (Leymus mollis), using long-read single-molecule real-time sequencing technology. Their genomes are assembled into four chromosomes and a mitochondrial genome with a total assembly size of 40.05 and 42.90 M, respectively. This resource should not only facilitate functional studies designed to better understand what makes the two Fusarium species such successful plant-beneficial fungi but should also reveal their genome evolution and adaptation.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A Novel Hexose Transporter ChHxt6 Is Required for Hexose Uptake and Virulence in Colletotrichum higginsianum.

Colletotrichum higginsianum is an important hemibiotrophic plant pathogen that causes crucifer anthracnose worldwide. To date, some hexose transporters have been identified in fungi. However, the functions of hexose transporters in virulence are not clear in hemibiotrophic phytopathogens. In this study, we identified and characterized a new hexose transporter gene named ChHxt6 from a T-DNA insertion pathogenicity-deficient mutant G256 in C. higginsianum. Expression profiling analysis revealed that six ChHxt genes, ChHxt1 to ChHxt6, exhibited specific expression patterns in different infection phases of C. higginsianum. The ChHxt1 to ChHxt6 were separately deleted using the principle of homologous recombination. ChHxt1 to ChHxt6 deletion mutants grew normally on PDA plates, but only the virulence of ChHxt4 and ChHxt6 deletion mutants was reduced. ChHxt4 was required for fungal infection in both biotrophic and necrotrophic stages, while ChHxt6 was important for formation of necrotrophic hyphae during infection. In addition, ChHxts were functional in uptake of different hexoses, but only ChHxt6-expressing cells could grow on all five hexoses, indicating that the ChHxt6 was a central hexose transporter and crucial for hexose uptake. Site-directed mutation of T169S and P221L positions revealed that these two positions were necessary for hexose transport, whereas only the mutation Thr169 caused reduced virulence and defect in formation of necrotrophic hyphae. Taken together, ChHxt6 might regulate fungal virulence by modulating the utilization of hexose.

High-quality chromosome-level genomes of Cucumis metuliferus and Cucumis melo provide insight into Cucumis genome evolution.

Cucumis metuliferus (African horned cucumber), a wild relative of Cucumis sativus (cucumber) and Cucumis melo (melon), displays high-level resistance to several important plant pathogens (e.g., root-knot nematodes and several viruses). Here, we report a chromosome-level genome assembly for C. metuliferus, with a 316Mb genome sequence comprising 29039 genes. Phylogenetic analysis of related species in family Cucurbitaceae indicated that the divergence time between C. metuliferus and melon was 17.8 million years ago. Comparisons between the C. metuliferus and melon genomes revealed large structural variations (inversions and translocations >1Mb) in eight chromosomes of these two species. Gene family comparison showed that C. metuliferus has the largest number of resistance-related nucleotide-binding site leucine-rich repeat (NBS-LRR) genes in Cucurbitaceae. The loss of NBS-LRR loci caused by large insertions or deletions (indels) and pseudogenization caused by small indels explained the loss of NBS-LRR genes in Cucurbitaceae. Population structure analysis suggested that C. metuliferus originated in Zimbabwe, then spread to other southern African regions where it likely underwent similar domestic selection as melon. This C. metuliferus reference sequence will accelerate the understanding of the molecular evolution of resistance-related genes and enhance cucurbit crop improvement efforts.

Multi-Gene Phylogeny and Morphology Reveal Haplohelminthosporium gen. nov. and Helminthosporiella gen. nov. Associated with Palms in Thailand and A Checklist for Helminthosporium Reported Worldwide.

Palms (Arecaceae) are substrates for a highly diverse range of fungi. Many species are known as saprobes and many are important plant pathogens. Over the course of our studies of micro-fungi from palms in Thailand, two new taxa were discovered. Morphological characteristics and phylogenetic analyses of combined ITS, LSU, SSU, and tef1-α sequence data revealed their taxonomic positions within Massarinaceae. There are currently ten genera identified and accepted in Massarinaceae, with the addition of the two new genera of Haplohelminthosporium and Helminthosporiella, that are introduced in this paper. Each new genus is provided with a full description and notes, and each new taxon is provided with an illustration for the holotype. A list of identified and accepted species of Helminthosporium with morphology, host information, locality, sequence data, and related references of Helminthosporium reported worldwide is provided based on records in Species Fungorum 2021. This work provides a micro-fungi database of Haplohelminthosporium, Helminthosporiella, and Helminthosporium which can be modified and validated as new data come to light.

Exploring the Biological and Molecular Characteristics of Resistance to Fludioxonil in Sclerotinia sclerotiorum From Soybean in China.

Sclerotinia sclerotiorum is one of the most damaging and economically important necrotrophic plant pathogens, infecting more than 400 plant species globally. Although the phenylpyrrole fungicide fludioxonil has high activity against S. sclerotiorum, reports indicate that there is also substantial potential for the development of fungicide resistance. However, the current study investigating five fludioxonil-resistant laboratory mutants found a significant fitness cost associated with fludioxonil resistance resulting in significantly (P < 0.05) reduced mycelial growth and sclerotia formation on potato dextrose agar as well as significantly (P < 0.05) lower pathogenicity on detached tomato leaves, with one mutant, LK-1R, completely losing the capacity to cause infection. In addition, all of the fludioxonil-resistant mutants had significantly (P < 0.05) increased sensitivity to osmotic stress (0.5 M of potassium chloride and 1.0 M of glucose), which is consistent with the proposed fludioxonil target sites within the high osmolarity glycerol stress response mitogen-activated protein kinase (HOG1-MAPK) signaling transduction pathway. Sequence analysis of six genes from this two-component pathway, including SsHk, SsYpd, SsSk1, SsSk2, SsPbs, and SsHog, revealed several mutations that may be associated with fludioxonil resistance. For example, six separate point mutations were found in SsHk that led to changes in the predicted amino acid sequence, including A136G, F249V, G353A, E560K, M610K, and K727R. Similarly, SsPbs had three mutations (D34G, S46L, and L337E), SsSk1 and SsYpd had two (S53G and A795V for SsSk1, and E67G and Y141H for SsYpd), and SsHog and SsSk2 had one each (V220A and S763P, respectively). To our knowledge, these constitute the first reports of amino acid changes in proteins of the HOG1-MAPK pathway being associated with fludioxonil resistance in S. sclerotiorum. This study also showed a positive cross-resistance between fludioxonil and dimethachlone and procymidone, but none with tebuconazole or carbendazim, indicating that the inclusion of tebuconazole within an integrated pest management program could reduce the risk of fludioxonil resistance developing in field populations of S. sclerotiorum and ensure the sustainable production of soybeans in China into the future.

Optimization of Growth Conditions of Citrus Anthracnose Agent Colletotrichum gloeosporioides Isolates in Tunisia

Background: Colletotrichum gloeosporioides is important plant pathogens on a wide range of plant hosts such as citrus causing pre- or post-harvest infections as anthracnose, post-bloom fruit drop, tearstain and stem-end rot on fruit, or wither-tip of twigs. Method: The optimization of growth conditions of this pathogen was performed (solid media, temperature, pH and water potential under laboratory experiments). Results: Our results revealed that the maximum radial growth of C. gloeosporioides was recorded on SDA medium. All isolates were able to grow on PDA at temperatures of 15 and 30°C (over 0.7cm/day). Optimal growth radial was recorded at pH 5, 6, 7 and 8. Similar responses were obtained with both salt types, but, in general, C. gloeosporioides was more tolerant to KCl than NaCl. Conclusion: Studies of cultural, morphological traits of the pathogen are prominent to understand the response of the pathogen in different environmental and nutritional conditions.

Genomic and Metabolomic Analysis of the Potato Common Scab Pathogen Streptomyces scabiei.

Streptomyces scabiei is a key causative agent of common scab disease, which causes significant economic losses to potato growers worldwide. This organism produces several phytotoxins that are known or suspected to contribute to host–pathogen interactions and disease development; however, the full metabolic potential of S. scabiei has not been previously investigated. In this study, we used a combined metabolomic and genomic approach to investigate the metabolites that are produced by S. scabiei. The genome sequence was analyzed using antiSMASH and DeepBGC to identify specialized metabolite biosynthetic gene clusters. Using untargeted liquid chromatography-coupled tandem mass spectrometry (LC-MS2), the metabolic profile of S. scabiei was compared after cultivation on three different growth media. MS2 data were analyzed using Feature-Based Molecular Networking and hierarchical clustering in BioDendro. Metabolites were annotated by performing a Global Natural Products Social Molecular Networking (GNPS) spectral library search or using Network Annotation Propagation, SIRIUS, MetWork, or Competitive Fragmentation Modeling for Metabolite Identification. Using this approach, we were able to putatively identify new analogues of known metabolites as well as molecules that were not previously known to be produced by S. scabiei. To our knowledge, this study represents the first global analysis of specialized metabolites that are produced by this important plant pathogen.

Phytochemical analysis and antimicrobial activity of an endophytic Fusarium proliferatum (ACQR8), isolated from a folk medicinal plant Cissus quadrangularis L.

The endophytic fungi are an important source of bioactive natural products. This study includes evaluation of antimicrobial activity and phytochemical analysis of endophytic fungus ACQR8, recovered from the root tissues of a folk medicinal plant Cissus quadrangularis L., which was identified as Fusarium proliferatum by microscopic and molecular methods. The fungus showed broad spectrum antibacterial and antifungal activities against variety of important plant and human pathogens. To the best of our knowledge, this is the first study to record antifungal activity of any endophytic Fusarium sp. against phytopathogens Rhizoctonia solani, Fusarium oxysporum and the dermatophytes Trichophyton mentagrophyte and Microsporum gypseum. The minimum inhibitory concentration of the crude extract ranged from 40–120 µg/ml against pathogenic bacteria and 0.2–2.5 mg/ml against fungal pathogens. Besides, this study probably is also the first to document the phytochemical constituents of an endophytic Fusarium sp. Phytochemical testing and GC-MS (gas chromatography- mass spectrometry) analysis revealed the presence of fatty acids and its derivatives, phenolics, terpenoids and unsaturated alkenes in the crude extract which scientifically validates the highly promising antibacterial and antifungal activity exhibited by endophytic isolate ACQR8.

Nep1-like proteins as a target for plant pathogen control.

The lack of efficient methods to control the major diseases of crops most important to agriculture leads to huge economic losses and seriously threatens global food security. Many of the most important microbial plant pathogens, including bacteria, fungi, and oomycetes, secrete necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs), which critically contribute to the virulence and spread of the disease. NLPs are cytotoxic to eudicot plants, as they disturb the plant plasma membrane by binding to specific plant membrane sphingolipid receptors. Their pivotal role in plant infection and broad taxonomic distribution makes NLPs a promising target for the development of novel phytopharmaceutical compounds. To identify compounds that bind to NLPs from the oomycetes Pythium aphanidermatum and Phytophthora parasitica, a library of 587 small molecules, most of which are commercially unavailable, was screened by surface plasmon resonance. Importantly, compounds that exhibited the highest affinity to NLPs were also found to inhibit NLP-mediated necrosis in tobacco leaves and Phytophthora infestans growth on potato leaves. Saturation transfer difference-nuclear magnetic resonance and molecular modelling of the most promising compound, anthranilic acid derivative, confirmed stable binding to the NLP protein, which resulted in decreased necrotic activity and reduced ion leakage from tobacco leaves. We, therefore, confirmed that NLPs are an appealing target for the development of novel phytopharmaceutical agents and strategies, which aim to directly interfere with the function of these major microbial virulence factors. The compounds identified in this study represent lead structures for further optimization and antimicrobial product development.